Thermocyclers and sample vessels are employed for the automated performance of the polymerase chain reaction (PCR). The process of deoxyribonucleic acid (DNA) amplification with PCR has become one of the most utilized techniques in molecular biology and conducting thermal cycling protocols is paramount to the technique. Various automated instruments to perform PCR thermocycling have been described in literature and are commercially available from numerous manufacturers.
PCR thermocycling instruments can generally be represented by three major classifications:                1) Conventional heat block cyclers which employ one or more heating/cooling apparatuses in contact with a thermally conductive block wherein PCR sample vessels are contained,        2) Capillary thermocyclers in which samples are contained within cylindrical glass or plastic capillaries which are exposed to convective heat transfer on their exterior, and        3) Microfabricated thermocyclers in which PCR samples are contained within etched, milled, or molded micrometer-scale structures and thermal cycling is achieved by different heat transfer methods such as resistive heating.        
All PCR thermocyclers seek to perform the temperature cycling necessary to facilitate the repeated PCR steps of denaturation, annealing, and elongation each of which generally occurs at different temperatures. As such, thermocycler performance is primarily based upon the thermocycler heating and cooling rates to reach these desired temperatures and by the hold time required for the heat to conduct to/from the PCR sample edge to the sample center. A high-performance thermocycler will rapidly change temperatures due to optimal thermocycler design and the high-performance thermocycler will have minimal denaturation, annealing, and elongation hold times due to optimal sample vessel design. The combined effect of temperature ramp rates and temperature hold times is what is critical to the performance of the instrument.
Exemplary instruments and apparatus employed for the performance of PCR thermocycling are disclosed in U.S. Pat. No. 6,556,940 to Tretiakov et al, U.S. Pat. No. 5,455,175 to Wittwer et al, U.S. Pat. No. 6,472,186 to Quintanar et al, U.S. Pat. No. 5,674,742 to Northrup et al, U.S. Pat. No. 5,475,610 to Atwood et al, U.S. Pat. No. 5,508,197 to Hansen et al, U.S. Pat. No. 4,683,202 to Mullis, U.S. Pat. No. 5,576,218 to Zurek et al, U.S. Pat. No. 5,333,675 to Mullis et al, U.S. Pat. No. 5,656,493 to Mullis et al, U.S. Pat. No. 5,681,741 to Atwood et al, U.S. Pat. No. 5,795,547 to Moser et al, U.S. Pat. No. 7,164,077 to Venkatasubramanian et al, U.S. Pat. No. 6,657,169 to Brown et al, U.S. Pat. No. 5,958,349 to Petersen et al, U.S. Pat. No. 4,902,624 to Columbus et al, U.S. Pat. No. 5,674,742 to Northrup et al, U.S. Pat. Nos. 6,734,401, 6,889,468, 6,987,253, 7,164,107, and 7,435,933 each to Bedingham et al, WO 98/43740, DE 4022792, WO/2005/113741, Northrup, M. Allen, et al, “A Miniature Integrated Nucleic Acid Analysis System”, Automation Technologies for Genome characterization, 1997, pp. 189-204, Wittwer, Carl T., et al, “Minimizing the Time Required for DNA Amplification by Efficient Heat Transfer to Small Samples”, Anal. Chem. 1998, 70, 2997-3002, and Friedman, Neal A., et al, Capillary Tube Resistive Thermal Cycling”, The 7th International Conference on Solid-State Sensors and Actuators, 924-926.
While each instrument design has its own benefits, all are subject to certain disadvantages. Heat block thermocyclers can generally handle a large number of samples with volumes of approximately 20-200 μl each. The conically shaped sample vessels used in most block cyclers are particularly advantageous for loading and unloading the sample mixtures by manual or automated pipettors. By using thermoelectric modules (Peltier devices) to provide heat pumping to the block, these thermocyclers require only electrical power to operate. However, these devices suffer from slow ramp rates and long minimum temperature hold times; usually requiring 1-3 hours to complete standard 30-cycle PCR protocols. The slow speed of these devices is generally attributable to the large thermal mass of the heat block, the use of thermoelectric modules on only one side of the heat block, the large wall thickness and poor thermal conductivity of the sample vessel, and the internal thermal resistance of the sample mixture itself.
To overcome slow ramp rates, some designs employ glass capillaries, such as disclosed in U.S. Pat. No. 5,455,175 to Wittwer et al, U.S. Pat. No. 6,472,186 to Quintanar et al, WO/2005/113741, and Friedman et al Capillary Tube Resistive Thermal Cycling”, The 7th International Conference on Solid-State Sensors and Actuators, 924-926. The glass capillaries provide a higher surface area to volume ratio and greater thermal conductivity than the conical sample vessels used in heat block thermocyclers, thereby creating the capability for rapid thermocycling. Hot-air thermocyclers using glass capillaries as disclosed in U.S. Pat. No. 5,455,175 to Wittwer et al, eliminate the thermal mass of heat blocks, but have relatively poor convection heat transfer properties. Improving on this idea, PCR using pressurized gas has been accomplished in a matter of minutes as disclosed in U.S. Pat. No. 6,472,186 to Quintanar et al and WO/2005/113741. However, as most molecular biology labs do not have readily available high pressure air, the application of pressurized gas devices is inconvenient and limited for many users. Also, glass capillaries are known to be fragile, more expensive, and require additional steps to load and unload the sample mixtures.
Microfabricated thermocyclers, as disclosed for example in U.S. Pat. No. 5,674,742 to Northrup et al, incorporate similar high surface area to volume ratios through the use of etched structures, usually in glass or silicon. While capable of fast thermocycling and integration with other laboratory techniques by the use of microfluidics, the manufacturing cost associated with these thermocyclers is high. As with glass capillaries, loss of enzyme activity and absorption of DNA onto the vessel surface are also problematic; and a carrier protein (e.g. bovine serum albumin) is recommended to reduce these undesired aspects. Additionally, these thermocyclers are usually limited to small reaction volumes on the order of a few microliters or less which is too small of a volume for many medically relevant PCR techniques.
Several advances have been made in the performance of block thermocyclers over the past decade. These are generally attributed to the use of thin-walled sample vessels with low thermal resistance as disclosed in U.S. Pat. No. 5,475,610 to Atwood et al, and low thermal mass sample blocks as disclosed in U.S. Pat. No. 6,556,940 to Tretiakov et al. Despite these advances, PCR cycling times and maximum reaction volumes for normal temperature protocols are far from optimal. In the apparatus of U.S. Pat. No. 6,556,940 Tretiakov et al, a rapid heat block thermocycler has a similar arrangement of components to conventional heat block cyclers. However, the Tretiakov et al instrument achieves fast thermocycling through the use of: 1) a low profile, low thermal mass, and low thermal capacity heat block, 2) at least one thermoelectric module, and 3) ultra-thin wall sample wells. This thermocycler can achieve much faster ramp rates than typical heat block cyclers; with PCR being capable of being performed in 10-30 minutes. Unfortunately, the reaction volumes are limited to 1-20 μL. Tretiakov et al has addressed two of the major handicaps of traditional heat block cyclers by reducing the thermal mass of the heat block and reducing the thermal resistance (i.e. wall thickness) of the sample vessel. However, the internal thermal resistance of the sample itself still limits the speed of the instrument. With the use of a conical shaped well, increases in reaction volumes changes the surface area to volume ratio and thus the internal thermal resistance becomes of greater significance. Therefore, larger volumes in the Tretiakov et al instrument would require longer hold times (and thereby increase run time) to enable the internal regions of the sample to reach proper temperatures needed for efficient PCR. The reaction volume is thus limited by Tretiakov et al to 20 μL for rapid PCR protocols. Additionally, larger volumes imply an increase in block height which leads to a larger heat block and thermal mass. Alternatively, a large vessel radius would increase internal thermal resistance.
U.S. Pat. No. 5,958,349 to Petersen et al discloses a sample vessel and thermocycler with abbreviated cycle times when compared to traditional block cyclers. The instrument takes advantage of a sample vessel with two major opposing faces through which the heat transfer primarily occurs. The sample vessel has a plurality of minor faces which join the major faces, a sample port, and a triangular shaped bottom that is optically advantageous. Sample heating is achieved through the use of heating elements in contact with the major faces; cooling is done by a chamber surrounding both the vessel and heating elements. The Petersen et al reaction vessel has a thermal conductance ratio of major to minor faces of at least 2:1. Petersen et al may employ different materials for the faces or different thicknesses, with the major faces having a higher conductance that allows for geometry modification of the vessel while still maintaining the thermal conductance ratio. This allows for the surface area ratio of major to minor faces to be less than 2:1, and subsequently condones a relatively large through thickness dimension (perpendicular to the heat transfer apparati). A high discrepancy (i.e. 10:1) of thermal conductances of the major to minor faces is allowed. A characteristic time is needed to transfer heat from the sample exterior to the interior regions to facilitate efficient PCR throughout the entire reaction mixture. By specifying a thermal conductance ratio and allowing large internal distances, the sample mixture itself can be rate-limiting. The internal thermal resistance of the sample mixture and its effect on the thermal kinetics of the system are overlooked by Petersen et al. In contrast, the sample vessel thermal path length was considered in U.S. Pat. No. 4,902,624 to Columbus et al. However, the design complexity of the sample vessel channels and reaction chamber proposed by Columbus et al are detrimental to heat transfer and are relatively costly to implement.
Many thermocyclers, especially heat block cyclers, use thermoelectric modules (Peltier devices) to facilitate temperature cycling. The sample vessel geometry dictates that a heat block which is complementary to the conical sample vessels be present between the thermoelectric module and the sample vessel. This heat block adds thermal mass to the system and slows cycling performance. Some in the art, such as U.S. Pat. No. 6,556,940 to Tretiakov et al, and U.S. Pat. Nos. 6,734,401, 6,889,468, 6,987,253, 7,164,107, and 7,435,933 each to Bedingham et al disclose the use of at least one thermoelectric module. Generally, multiple thermoelectric module configurations are 1) in stackable configurations to achieve higher temperature differences between the outside faces or 2) to create temperature differences among sample vessels as with temperature gradient cyclers. Multiple modules may also be used in multiple heat block cyclers that can run separate thermocycler protocols simultaneously. However, the multiple modules are used only on one side of the heat block (generally the bottom side).
Conventional heat block instruments would not substantially benefit from the presence of a thermoelectric module on the top surface of the heat block. A top thermoelectric module cannot practically be employed in conventional block cyclers as is especially evident in most commercially available block cyclers in which heated lids are utilized to reduce detrimental sample evaporation/condensation. The heated lids do manipulate the temperature of a portion of the sample vessel but only in an isothermal manner and there is a significant insulating air gap present between the lid and the sample mixture making it unfeasible to conduct temperature cycling at this lid surface. Therefore, the heated lid serves a limited function and does not directly participate in the temperature cycling protocol to achieve PCR.
The thermocycler apparatus of the present invention has a unique arrangement of thermocycler components and sample vessels that enable rapid temperature cycling. The use of two or more thermoelectric devices placed in spatial opposition to one another yields very dense heat pumping to samples within the interior space. In embodiments of the present invention, thirty cycles of PCR can be completed in mere minutes, significantly less than any other solid-state apparatus and on par with the fastest of compressed air thermocyclers.
Another aspect of the present invention that enables rapid PCR is the use of specifically designed sample vessels. Not all sample vessels are capable of rapid temperature cycling even with thin walls. Efficient PCR demands that all regions of the sample reach the desired set point temperatures at each PCR step. Thus, outer regions of the reaction mixture must be held at the desired temperature whilst the interior regions reach the desired temperature. For example, conical tubes used in standard heat block cyclers recommend hold times of about 30 seconds even though PCR steps (such as denaturation and annealing) are nearly instantaneous events. Despite their advantages for sample loading and larger volumes, standard conical PCR tubes are not amenable to rapid PCR. The samples vessels disclosed in the present invention are marked by several key characteristics. The sample vessels employed in the present invention are easy to load similar to standard conical PCR tubes when outside of the thermocycler, yet can be used for rapid PCR by limiting the thickness dimension critical to temperature cycling when inserted into the thermocycler. Most importantly, larger reaction volumes can be processed without any substantial increase in PCR runtimes, a consequence of the novel design of the invention. In comparison to the vessel of U.S. Pat. No. 5,958,349 to Petersen et al., the sample vessel of the present invention need not have a plurality of minor faces. The sample vessel of the present invention may include cylindrical regions that are continuous. Instead of defined edges as in Petersen et al., the continuity and deformability of the sample vessels of the present invention facilitates improved thermal contact. Also, rapid PCR is not reliant on specifying a thermal conductance ratio, but rather the heat transfer kinetics from outer sample regions closer to the heat source (or sink) to the inner regions. In contrast to the sample vessel of U.S. Pat. No. 4,902,624 to Columbus et al., the sample vessel of the present invention is much simpler in design and thus manufacture, while at the same time performing at much higher speeds. The deformable and accessible nature of sample vessels disclosed herein offer unique advantages for sample loading and thermal contact than non-deformable sample vessels such as glass capillary and conical sample vessels.
Fourier's law of conduction and the thermal conductance of the system (conductivity divided by the material thickness) have been referenced in the design of many PCR thermocyclers and sample vessels. While thermal conductance is a relevant design parameter for steady state heat transfer, the temperature cycling of PCR is a dynamic process. As such, it is more apt to include the time dependency through the application of the heat diffusion equation, a parabolic partial differential equation that is derived from Fourier's law of conduction and the conservation of energy:
            ∂      T              ∂      t        =            κ      ⁢                        ∇          2                ⁢        T            ⁢                          ⁢      where      ⁢                          ⁢      κ        =          k              ρ        *                  C          p                    The change in temperature (T) over time (t) depends upon the thermal diffusivity (κ) and the Laplacian of the temperature (∇2T). Thermal diffusivity includes the thermal conductivity (k) and the thermal mass (ρ*Cp) where p is the material density and Cp is the heat capacity. The Laplace operator is taken in spatial variables of the physical system. The unassuming heat equation is quite powerful when applied to PCR thermocycling and its solution can be found for different physical systems by a variety of analytical or numerical methods. Qualitatively, one can extract the key design parameters directly from the above equation. To maximize speed, the thermal conductivity should be large while the thermal mass small. A small thermal mass is achieved by keeping the spatial dimension to a minimum.
In embodiments of the invention, the heat diffusion equation is applied to all regions, yielding a system of coupled equations. The temperature behavior should be elucidated not only for regions on the exterior of the vessel and the vessel wall, but also for the sample mixture itself. During PCR temperature cycling, overshoot of the denaturation temperature is undesirable because of thermal damage to the DNA and loss of enzyme activity. An undershoot of the annealing temperature is harmful to PCR because of possible misannealing events. Therefore, a characteristic time is employed to allow for proper temperatures to occur throughout the sample while not allowing significant overshoots or undershoots at the sample mixture exterior. Since the thermal diffusivity and mass of the sample mixture and temperature set points are dictated by the PCR process, limiting one of the spatial dimensions of the sample mixture is the best method to facilitate rapid temperature cycling. By application of these fundamental principles of heat transfer, the present invention provides a geometry and arrangement of components and sample vessel design for rapid PCR thermocycling. By limiting the internal distance of the sample mixture and placing thermoelectric modules in intimate proximity to the sample vessel, the present invention achieves rapid sample thermocycling and efficient PCR. Additionally, the arrangement of thermoelectric modules according to the present invention not only reduces the distance from the heat transfer sources to the reaction mixture, it increases the effective heat pumping density available to the samples.